ADP-ribosylation factors (ARFs) are about 20 Kda guanine nucleotide- binding proteins initially identified by their ability to enhance in vitro cholera toxin-catalyzed ADP-ribosylation and subsequently shown to participate in vesicular transport in the Golgi and other cellular compartments. By CDNA and genomic cloning, at least six mammalian ARFs were identified. Brefeldin A (BFA) disrupts Golgi membranes, and inhibits binding of soluble high molecular weight proteins to Golgi fractions. We examined the effects of BFA on binding of ARFs 1,3, and 5 to a Golgi fraction in the presence of an ATP-regenerating system and a fraction of soluble, high molecular weight, accessory proteins (SAP), presumably containing complexes identified by others as coatomers that are involved in vesicular transport. ARF binding in all instances was dependent on GTPgammaS and increased by the ATP-regenerating system. Binding of ARFs 1, and 3, but not ARF 5, was enhanced by SAP. BFA inhibited the SAP-dependent, but not the SAP-independent , binding of ARFs 1 and 3. It had no effect on the increment in binding produced by an ATP-regenerating system. B36, an inactive derivative of BFA, did not inhibit SAP-dependent binding of ARFs 1 and 3. Binding of ARF 5, which was SAP-independent, was not affected by BFA. These observations are consistent with the conclusion that mammalian ARFs differ in their dependence on accessory proteins for interaction with Golgi and, perhaps, other cellular membranes, and that BFA specifically inhibits SAP- dependent ARF binding.